Department of Medicine, University of California, San Francisco, California.

5

Department of Pathology, University of California, San Francisco, California; stephen.nishimura@ucsf.edu.

Abstract

Small airway fibrosis is a major pathological feature of chronic obstructive pulmonary disease (COPD) and is refractory to current treatments. Chronic inflammatory cells accumulate around small airways in COPD and are thought to play a major role in small airway fibrosis. Mice deficient in α/β T cells have recently been shown to be protected from both experimental airway inflammation and fibrosis. In these models, CD4+Th17 cells and secretion of IL-17A are increased. However, a pathogenic role for IL-17 in specifically mediating fibrosis around airways has not been demonstrated. Here a role for IL-17A in airway fibrosis was demonstrated using mice deficient in the IL-17 receptor A (il17ra) Il17ra-deficient mice were protected from both airway inflammation and fibrosis in two different models of airway fibrosis that employ COPD-relevant stimuli. In these models, CD4+ Th17 are a major source of IL-17A with other expressing cell types including γδ T cells, type 3 innate lymphoid cells, polymorphonuclear cells, and CD8+ T cells. Antibody neutralization of IL-17RA or IL-17A confirmed that IL-17A was the relevant pathogenic IL-17 isoform and IL-17RA was the relevant receptor in airway inflammation and fibrosis. These results demonstrate that the IL-17A/IL-17 RA axis is crucial to murine airway fibrosis. These findings suggest that IL-17 might be targeted to prevent the progression of airway fibrosis in COPD.

Lung and airway inflammation, and airway wall fibrosis induced by cigarette smoke in combination with intranasal polyinosinic:polycytidylic acid is dependent on IL-17RA/IL-17A. WT or il17ra-deficient (il17ra−/−) mice were treated with cigarette smoke (CS) in combination with polyinosinic:polycytidylic acid (PIC) or room air (RA) as a control (A–E). After 14 days, bronchoalveolar lavage (BAL) was performed and total cell counts (A), macrophage (B), or neutrophil numbers (C) were assessed. Morphometric analysis of airway inflammation (D) or airway wall fibrosis was performed from histological sections (E). WT mice treated with either RA or CS+PIC with isotype, or anti-IL-17RA (F–J), or anti-IL-17A (K–O) underwent the same BAL (F–H, K–M) and morphometric analysis (I, J, N, O), as above. Experiments were repeated a minimum of 3 times with at least 3 mice/group. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

CD4 + IL-17-expressing cells (Th17) are significantly increased in the lung by exposure to CS+PIC. WT C57B/6 mice were exposed to CS+PIC and the lungs harvested on day 21 after the initiation of smoke exposure. Lung immune cells were analyzed by multicolor flow cytometry. Cells were stained in a single panel for Gr1, NK1.1, B220, TCRβ, TCRγδ, CD8, CD4, IL17A secretion, and INFγ secretion (A–I) or for CD3ε, CD90, CD127, Rorγt, IL17A, and INFγ (J–L). A: cells expressing Gr1, Nk1.1, or neither were gated and percent positive staining with IL-17A shown as total positive cell number in M. In B, Gr1, Nk1.1 double-negative cells were gated into Tcrγδ (D–F)- and Tcrβ (C)-positive populations, which were then gated into IFNγ-negative and IL-17A-positive populations in room air (E) compared with CS+PIC (F)-treated mice. IL-17A-positive cells were defined using pooled cells stained without surface capture for IL-17A (D). In C, Tcrβ-positive cells were gated into CD8- and CD4-positive populations. Shown in in G–J, Tcrβ, CD4+ or CD8 (not shown) cells were gated into IL-17A-positive and IL-17-negative populations, which were enumerated as shown in M. J: lung cells were gated into CD3ε and CD90-positive and -negative populations. Innate lymphoid cells (CD3ε neg, CD90 positive) were gated into CD127 positive/IL-17-negative (ILC1) and CD127-negative/IL-17A-positive (ILC3) populations in room air (K) and CS+PIC-exposed mice (L) and enumerated in M. IL-17A-positive cells were defined by pooled cells stained without IL-17A surface capture. IL-17A-positive ILC3 cells were also positive for Rorγt (not shown). In M, total numbers of IL-17A-expressing cell types (PMNs, NK, CD4, CD8, γδ, and ILC3) in room air (open bars) and CS+PIC-exposed (solid bars) mice (n = 3 in each group) are shown. *P < 0.05, **P < 0.01 as determined by Student’s t-test.